Sod translocation to restore habitats of the myrmecophilous butterfly Phengaris (Maculinea) teleius on former agricultural fields

Abstract In Europe, 50%–70% of former natural grassland area has been destroyed during the past 30 years due to land use changes, losses are expected to increase in the future. Restoration is thought to reverse this situation by creating suitable abiotic conditions. In this paper, we investigate the effects of sod translocation with specific vegetation to facilitate the restoration of a former intensive agricultural field into a wet meadow. First, starting conditions were optimized including modification of the local hydrology, removal of the fertilized topsoil, application of liming, and translocation of fresh clippings as a seed source. The second part aimed at restoring the habitat for the butterfly species Phengaris (Maculinea) teleius, one of the species that was especially affected by the loss of wet meadows. This species engages in a complex myrmecophilous relationship with one host plant, Sanguisorba officinalis, and one obligate host ant, Myrmica scabrinodis. We used sod translocation to create islands of habitat to promote host plant and host ant colonization. After 4 years following the restoration, we observed that plants spread from the transplanted sods to the surroundings. The vegetation composition and structure of the transplanted sods attracted colonization of Myrmica ants into the restored areas. Following the increase in vegetation cover and height, Myrmica ant colonies further spread into the restored areas. Therefore, sod translocations can be considered an effective restoration method following topsoil removal in the process of restoring wet meadows to provide a starting point for ant colonization and plant dispersion. With these findings, this paper contributes to the evidence‐based restoration of wet meadows on former agricultural fields, including complex interactions between invertebrates and their required ecological relationships.


| INTRODUC TI ON
Landscapes have been severely modified by changes in land use in Europe (Barrett et al., 2018;Newbold et al., 2015;Tscharntke et al., 2012;Warren et al., 2021). Landscapes consisting of a mosaic of natural and seminatural habitat types shaped by traditional low-intensity agricultural practices have changed into landscapes of large and intensively used agricultural fields or they are encroached by shrubs after abandonment of agriculture (Craioveanu et al., 2021;Loos et al., 2021). In Europe, 50%-70% of former grassland area has been destroyed during the past 30 years (Török et al., 2021). In addition, the highest proportion of habitats with an unfavorable and deteriorating conservation status in the European Union is found in natural grasslands (European Commission, 2015, 2021. European seminatural grasslands supporting a high biological diversity are assumed to have lost at least 90% of their former area during the last century (Cosentino & Schooley, 2018;WallisDeVries et al., 2002). Moreover, climate and land use changes are severe threats to the future of particularly wet grasslands and related species. Landscape homogenization resulted in the fragmentation or loss of habitat for many populations of plant and animal species found in grasslands. The distances between suitable patches have increased and even when grassland habitats are restored, many species are not able to colonize them without support (Bakker & Berendse, 1999). These problems are more evident for species with strict habitat requirements, with a limited distribution, or with low dispersal capabilities (Büchi & Vuilleumier, 2014;Fourcade et al., 2021). Therefore, there is a wide interest in restoring grassland habitats of vulnerable species, however, most restoration projects are based only on vegetation targets or single species while the integration of the whole ecosystem is missing (Goreth et al., 2021;Török et al., 2021).
The first step to restore grasslands on formerly intensively used agricultural fields is the reestablishment of suitable abiotic conditions such as restoring a natural water regime or removing nutrient-rich top soils (Klimkowska et al., 2007;Zedler & Miller, 2018). Even after solid preparation of the starting conditions, natural colonization of many species to the restored habitats cannot be taken for granted. For certain groups of organisms, translocation offers a possibility for successful colonization into the restored new habitats. For example, plant species have been moved to newly established patches of habitat by transportation of seeds or young plants (Donath et al., 2007;Goreth et al., 2021;Török et al., 2021;Vitt et al., 2016;Wagner et al., 2021), preferably from sites with a common genetic background (Höfner et al., 2021). Moreover, birds, mammals, amphibians, and some butterflies are also translocated as soon as the new patches have developed into suitable habitats (Ferrer et al., 2017;Germano & Bishop, 2009;Wynhoff, 1998). However, within restoration projects, specific taxonomic groups are over-represented (Donaldson et al., 2016;Kollmann et al., 2016;Martín-López, 2009) with birds, mammals, and vascular plants being the main target, while invertebrates are underrated (Kollmann et al., 2016). Recently, the necessity of restoring habitats with a broader view, embracing interactions between species, has been stressed, including trophic interactions, as pollination, soil fertility, or bio-engineers (European Commission, 2021;Fraser et al., 2015;Kollmann et al., 2016). For instance, including interactions between species that have proven to be so-called ecosystem engineers, such as earthworms or ants could further enhance the success of habitat restoration (Lavelle et al., 2016).
Grassland butterflies are some of the most affected organisms of the changes in natural grasslands (Van Swaay et al., 2015;Warren et al., 2021). This group of insects can be used as indicators of grassland status and effectiveness of applied restoration methods (Musters et al., 2013;Van Swaay et al., 2015). A meta-analysis of prairie grassland restoration showed that butterfly abundance increased more than bee abundance, especially with multiple restoration methods applied, and older restorations showed the strongest improvements (Sexton & Emery, 2020). However, complex interactions between species such as the case of Phengaris (Maculinea) butterflies which have a parasitic relation with ants were not included (Sexton & Emery, 2020). The only example of a successful restoration of these complex host-parasite interactions in butterflies is found in the United Kingdom where limestone grassland habitats of the butterfly Phengaris (Maculinea) arion have been restored (Thomas et al., 2009). In 1979, the first reintroductions of M. arion started and 30 years later, ca. 40 sites have been colonized by the butterfly thanks to the strong emphasis on the relationship between this butterfly and its local host ant (Thomas et al., 2009). Therefore, restoration projects aiming to improve the conservation status of butterflies with complex host-parasite interactions should have a broader view and focus on their interactions. This paper describes the habitat restoration within the LIFE+ project "Blues in the marshes" for a butterfly species with a comparable life cycle to M. arion but restricted to wet fen meadows, where the interactions of invertebrates with the grassland ecosystem is the main focus (Natuurmonumenten, 2018).
The project aims to enlarge the wet meadow habitat of the butterfly species Phengaris (Maculinea) teleius (from now on M. teleius) ( Figure 1) by creating suitable conditions in the surrounding areas for the butterfly population to expand (Natuurmonumenten, 2018).
In the Netherlands, only one population of this rare butterfly exists after being reintroduced in 1990 (Wynhoff, 1998), but it has been confined to only 3 ha for more than 25 years. The young caterpillars of the butterfly are monophagous on the host plant

Restoration ecology
Sanguisorba officinalis, which is abundant on moist fen meadows (Thomas, 1984). After 3 weeks feeding on the plant, the caterpillar is adopted by the host ants and taken into the ant nest where it hibernates (Witek et al., 2010). There are several host ants for M. teleius across Europe (Tartally et al., 2019). However, in the Netherlands, the caterpillars survive only in nests of Myrmica scabrinodis and usually on meadows with only this single species present (Van Langevelde & Wynhoff, 2009). Since both host plants and host ants are needed for its survival, the restoration process is necessarily based on the requirements for these two host species to provide suitable habitat for the butterfly. Thus, the major challenge to achieve the restoration of M. teleius habitat, is to reach an adequate density of both host plants and host ant nests to enable survival after colonization of the butterfly. Another problem of this system is that both hosts have low propensities to colonize new areas through dispersal (Elmes et al., 1998;Matus et al., 2003). Therefore, in early stages of the restoration, the host plant was translocated with fresh clippings as seed source from nearby wet fen meadow vegetation and with sod translocations . Sod translocations consist of a transplant of the target vegetation from wet meadows into the restoration area, which are expected to also increase the probability of ant colonization .
In this study, we investigate the effects of sod translocations of the target vegetation to the restoration areas on the establish- We hypothesized that sod translocations accelerate the vegetation development in the restoration area (hypothesis 1). The transplanted sods are expected to promote M. scabrinodis colonization and establishment. Our hypothesis was that Myrmica ants colonize the restoration areas starting in the translocated sods as these sods are assumed to be islands of suitable habitat for them (hypothesis 2).
Over the course of time, the vegetation is expected to get denser and taller, further promoting the distribution of M. scabrinodis in the restoration areas (hypothesis 3).

| Study site
A restoration project was carried out in the Natura 2000 area "Vlijmens Ven, Moerputten and Bossche Broek" (931 ha), located south of the city of 's Hertogenbosch, the Netherlands ( Figure 2). The core site Moerputten (115 ha) consists of moist meadows and wet forests. In the past, the surrounding area was dominated by intensively used agricultural fields and cattle pastures (Wynhoff, 1998;. The wet meadows in Moerputten provide the habitat for M. teleius which is restricted to one core population in this reserve. The restoration actions were described in detail earlier . The restoration areas were at distances from the butterfly population within the known long dispersal range (average 2 km, maximum 4.5 km) (Van Langevelde & Wynhoff, 2009). In the restoration areas, suitable abiotic conditions were restored in terms of basic seepage, water accessibility, removal of the fertilized soil, and the preservation of high winter water tables to maintain nutrient-poor conditions . The top 40 cm of phosphate-enriched soil on a total of 250 ha was excavated. The development of the target vegetation was facilitated by liming (1000 kg/ha) and transfer of freshly cut clippings on the excavated areas from the nearby nature reserve (Donath et al., 2007;Höfner et al., 2021;Hölzel & Otte, 2003;Matus et al., 2003;Török et al., 2011). Starting 1 year later, all restored meadows were mown yearly in summer. Finally, vegetation sods consisting of a transplant of suitable habitat for M. teleius were translocated from meadows in Moerputten (see details below and in Figure 2).

| Sod translocation experiment
The sod translocation experiment was conducted twice, the first  Figure S1h).
As a consequence of the weather conditions in both years of sod translocation (average temperature in both years of 11°C following a week of cold weather), Myrmica queens were assumed to hide deep in the soil (>10 cm deep) for hibernation (Kipyatkov & Lopatina, 1999) and hence not be translocated together with the sods. To test whether this assumption is true, we sampled ants three times during the second sod translocation in October 2016. The first time was right after lifting the sods, and we found worker ants under seven sods. Then we placed ant baits in the translocated sods after 1 week and after 10 months of the translocation, and we found worker ants in seven and five sods, respectively (I. Wynhoff, unpublished data). In only one sod, worker ants were found more than once and the rest of the captured ants were distributed randomly in each capture event. Thus, since we also did not find queens, we concluded that effectively ants were not translocated within the sods and that those found during the experiment (e.g., in 2014;  were colonizing from outside the topsoil removed area. In our study, sods were moved to sandy soil with sparse vegetation in different densities. At each patch, nine sods were placed in a 3 × 3 grid ( Figure 3, Figure S1g). In 2013, a distance of three meters was kept between the sods. Control plots of the same dimensions of the sods were established at the same distances around the sods (c-controls in Figure 3). It is expected that worker ants from the same colony could only be found in one sod or control; thus, the frequency F I G U R E 2 Study area. Natura 2000 nature reserve (purple line) with the core area of Moerputten and restored areas, Vlijmens Ven (VV) and Honderd Morgen (The Netherlands). Yellow polygons locate restored patches: VV1 to VV2, VV3 to VV7, HMD (=Honderdmorgensedijk Driehoekje), HOM (=Honderdmorgensedijk), TCG1 and TCG2 (=Tegenover Compensatiegebied) and CG1 and CG2 (=Compensatiegebied). The table below shows the restoration methods applied in different years during the research.
of ant occurrences within a patch was assumed to be independent of species' activity densities (Dahms et al., 2010). After the rapid colonization of the sods in 2014 , eight additional controls per patch were added at random locations of at least 10 m distance from the patch in 2015 (o-controls in Figure 3). The second translocation in 2016 (VV meadows) mainly serves to prove that effectively ants were not translocated with the sods, therefore, the second translocation data are not included in the analysis. All meadows with translocated sods were managed equally. In each year after sod translocation, they were mown in October/November. After cutting, the hay was left there for several days and then removed.

| Data collection
In July and August 2014 and 2016, 1 m 2 vegetation relevés were performed on the sods and the c-controls of the first sod translocation experiment in 2013 according to the Braun Blanquet method (Meijden & Bruinsma, 2007). All plant species were listed and their coverage was estimated. The Ellenberg values of nitrogen, moisture, and pH per relevé were calculated using the program Turboveg (Hennekens & Schaminée, 2001). Every year from 2014 until 2017, the vegetation structure was recorded including the cover of shrubs, herbs, mosses, total vegetation, dead organic matter (from now on DOM), and bare soil on all transplanted sods and controls. In addition, we measured the height of the vegetation using the Barkman stick method (Barkman, 1979;. In total, five measurements were taken per relevé and were averaged. The standard deviation (SD) was used as a proxy for variation in vegetation structure.
To collect data on ant presence in all patches, plastic pitfall tubes were placed (15 ml, Ø1.7 cm, 12 cm long) filled with fruit wine (mixture of raspberry, blackcurrant, cherry, 8.5% alcohol) in the soil in the middle of the plots, with the top of the tube level with the ground surface. Tubes were collected 24 h after positioning, covering all periods of daily activity of the ants. Baits were placed between mid-July and August every year. All ant species were identified using Boer (2010).

| Vegetation
First, differences in vegetation structure were analyzed to assess the effect of year of experiment and treatment (sods and c-controls for the 4 years of research and o-controls for the last 3 years) on different environmental variables that experienced changes. We performed beta regression models with a Beta distribution for the variables measured as a percentage (i.e., total vegetation cover, shrub cover, DOM cover, moss cover, and Sanguisorba cover), and GLMMs with a normal distribution for height variables (i.e., mean and Standard Deviation vegetation height) using Patch ID as random factor. For the beta regression models, we calculated the significance of each environmental variable by using a likelihood ratio test between models with and without a specific environmental variable.

| Ants
To test whether ants colonized the restoration areas starting in the translocated sods, we tested whether differences in the presence/ F I G U R E 3 Location of the nine transplanted sods (purple) and control relevés (c-controls: grey) within a patch. Ccontrols were placed randomly. Distance between the sods and c-controls (x) is 3 m for Honderd Morgen meadows and 6 m for Vlijmens Ven meadows. Black dots indicate ant baits. In 2015, an additional 8 controls were placed outside each patch at a distance of at least 10 m (o-controls: yellow).
absence of M. scabrinodis, Lasius niger, all Myrmica species and all ant species together were determined by the treatment, year of experiment and their interaction with a series of Generalized Linear Mixed Model (one GLMM analysis was done for each). In these GLMMs, we used a binomial distribution with logit link function and Patch ID as random factor.
In addition, another series of GLMM (with binomial distribution and logit link function) was performed to test which environmental variables affect the occurrence of ants in the restoration areas; we incorporated all environmental variables in the GLMMs (one factor included in each model) for the presence/absence of the ants. Environmental variables were standardized to compare the effect sizes of them. Here we used year of experiment as random effect, the highest estimated value of the coefficients to determine which variable explained ant presence best and the adjusted p-values according to Benjamini-Hochberg procedure using a false discovery rate of 10% for significant values (FDR = 0.1) (Benjamini & Hochberg, 1995). Additionally, to test the presence of L. niger on the

| Vegetation
All sods survived the transplantations of 2013 and subsequent years of the research. After the start of the experiment, the vegetation structure of all plots changed during the years of experiment and differences between treatments and locations were found as a consequence of vegetation development. We found significant differences in total vegetation cover, herb cover, shrub cover, bare soil cover, DOM cover, moss cover, mean, and SD of vegetation height, between the years of experiment and treatments, and Sanguisorba cover only between treatments (Table 1). Overall, the sods showed a higher total vegetation cover, higher vegetation height and more On the right-hand side, the c-controls are more scattered due to the lack of similarity between plots (Figure 4). The c-control plots were correlated with moss cover (ρ = .27, p < .001, df = 178), shrub cover

| Ants
Ten ant species were captured throughout the four investigated  Table 2). The presence/absence of all ant species found on the investigated plots was different between the treatments but not for the years of experiment since sods translocation (Table 2, Figure S5b).
We found effects of the changes in certain vegetation parameters on the ants (Table 3). The distribution and presence of M. scabrinodis were mostly correlated with the environmental variable total vegetation cover and bare soil cover showing the higher effect sizes resulting in higher probabilities of occurrence (Table 3, Figure 6a,b). These last two variables have opposite effects and were highly correlated (Pearson correlation, ρ = −.85, p < .001**, Figure S6). As the herb cover was correlated with the total vegetation cover (Pearson correlation, ρ = .87, p < .001**), it also showed a large impact on the presence of this ant. The mean vegetation height also was significant but its effect size was lower, with M. scabrinodis being more likely to occur in areas with taller vegetation (Figure 6c).
The cover of Sanguisorba was just significant for the presence of M. scabrinodis. Ellenberg values of nitrogen, moisture, and pH did not have an influence on the presence of M. scabrinodis, however those values were significant for the presence of L. niger (Table 3).
M. scabrinodis avoided areas where L. niger was present, suggesting a competition effect between both ant species. The year of excavation was significant for the presence of the ants; the more years passed since excavation the higher the probability of finding ants.
On the other hand, the presence of L. niger was correlated to fewer and different variables ( Table 3) The probability of L. niger 's occurrence slightly decreased with increasing total vegetation cover and decreased drastically with higher vegetation (Figure 6a,c), but was not affected by bare soil cover   (Table S7).

| DISCUSS ION
The loss of wet meadows has been dramatic in Europe during the last century, and nowadays, they are still threatened by climate and land use changes (Cosentino & Schooley, 2018;Joyce, 2014;WallisDeVries et al., 2002). In this paper, we investigated whether phases as lack of vegetation coverage, more moss cover, and high levels of nutrients (Smith et al., 2002;Zedler, 2000).
Here, we showed how the restoration area (sods) acquired already wet meadow characteristics over the study period, and the vegetation composition on the c-controls may shift toward the vegetation composition of the sods (Figure 4). Sod translocation leading to the simple proximity of the target vegetation community might help plant propagation and increase the likelihood of success to cover the area over time (Jansen et al., 2000;Matus et al., 2003).
Indeed, a remarkable number of 51 new species was found in a short   Table S2). Moreover, the accumulation of dead organic matter (DOM) is one consequence derived from vegetation development toward more mature ecosystem stages (Jansen et al., 1996).
DOM interacts with several conditions of the soil, physically and chemically, that link the soil biodiversity and ecosystem functions (Bot & Benites, 2005). With our restoration, DOM fluctuated over time, increasing only for c-controls (Table 1 and Table S2). Finally, the reduction of moss cover in the sods and c-controls, slight decrease of Ellenberg nitrogen value in sods and c-controls and the appearance of higher shrub coverage in the c-controls and (nonsignificant) in the o-controls are also signs of the development shifting away from primary phases over time (Middleton, 2018). In our experiment, the o-controls performed better than the c-controls. All these characteristics that we observed during the study period, indicate that the vegetation structure is moving toward a healthy wet meadow (Sammul et al., 2012;Zedler, 2000).
Our results showed that the sod translocation method enabled the host Myrmica ants to colonize new areas where they were initially absent due to the soil excavation. We validated that no queens were moved with the sod translocation, so colonization was dependent on external founders: young mated queens dispersing after their nuptial flights. The ants found in the sods came from outside the topsoil removed areas. M. scabrinodis generally avoids areas dominated by bare soil because the conditions are extreme, with high temperatures and drought during summer days, while moist conditions and moderate temperatures are kept stable by vegetation cover (Elmes et al., 1998;Trigos-Peral et al., 2018;. Therefore, if the vegetation cover around the sods was increasing, the ants could occupy those areas that offered their required ecological conditions of moisture and temperature (Elmes & Wardlaw, 1982;Procházka et al., 2011). Indeed, the different restoration methods applied, and in particular the sod translocations, allowed a fast colonization of M. scabrinodis ( Figure 5). These findings support our hypothesis 2. Only in 2016, the process was slowed down due to frequent heavy rains in the summer. As the area covered by vegetation as well as its height increased, the probability of occurrence of M. scabrinodis also increased ( Figure 6), supporting our hypothesis 3. Furthermore, to be able to build a nest, Myrmica ants need some support by plant material, such as roots or stems but L. niger is able to start a colony in a shallow nest without any support (Kipyatkov & Lopatina, 1999).  shows that S. officinalis cover increases the presence of M. scabrinodis but this is because both butterfly hosts, M. scabrinodis and S.
officinalis, have similar ecological requirements. Therefore, in the coming years the probability is high that they will continue to occur in each other's vicinity.
In the restored meadows, spatial separation of the two main ant species was found. The presence of bare soil negatively influences the presence of M. scabrinodis and facilitates the colonization and spreading of its main competitor, L. niger ( Figure 6). During colonization of new habitat, the presence of L. niger may obstruct the colonization of M. scabrinodis (Elmes et al., 1998). Indeed, we found a negative correlation between the two species, which suggests that the species exclude each other ( Table 3) for helpful recommendations that improved our manuscript.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare that are relevant to the content of this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data supporting this article are uploaded at the Dryad repository: https://doi.org/10.5061/dryad.1jwst qjz2.